Defining the role of HMGA2 in driving tRNA synthesis in pancreatic ductal adenocarcinoma

胰腺导管腺癌（PDA）是一种极其致命的疾病，5年生存率低于10%，到2030年可能成为癌症相关死亡的第二大原因。 我们知道胰腺癌是复杂的，因为患者对化疗的反应非常不同。为了理解这些差异，研究人员最近描述了两种主要亚型：经典/胰腺祖细胞和准间充质（QM）/基底/鳞状细胞。QM PDA亚型更具侵袭性，总体生存率最差。了解每种亚型的定义，它们的耐受性和耐药机制将有助于为这种毁灭性疾病确定新的靶向治疗。在研究这些问题时，Kugel实验室发现了一个惊人的发现，QM PDA亚型表达高水平的高迁移率族（HMG）蛋白A2（HMGA 2），并进化出了显著增加蛋白质翻译的机制。这提出了一个有趣的可能性，即HMGA 2的异常激活增加了蛋白质翻译，可能是PDA获得高度侵袭性QM表型的一种机制。本研究的目的是阐明HMGA 2在QM PDA中调节tDNA转录的作用，确定差异表达的tRNAs对蛋白质合成的作用，并最终探索HMGA 2在PDA亚型确定、启动和维持中的作用。因此，将利用癌症基因组图谱研究潜在的候选tRNA，将在HMGA 2（HMGA 2高/QM PDA）细胞系中针对HMGA 2低/经典PDA细胞系进行验证。将在经典或QM PDA细胞系中检测HMGA 2对tRNA表达的直接影响，这些细胞系分别通过tRNA测序工程化以过表达WT HMGA 2或通过siRNA沉默HMGA 2。HMGA 2是一种结构性转录因子。我们将通过对工程化PDA细胞系中与候选tRNA基因体结合的典型和非典型组蛋白进行ChIP分析，测定HMGA 2对染色质结构的影响，以诱导tDNA转录。我们将进行核糖体分析，以确定差异表达的tRNA引起的蛋白质合成的变化。最后，工程化细胞系将用于评价HMGA 2对QM标志物的影响，如增加的TP 63 DeltaN和间充质标志物表达、增加的染色质结合MYC水平和降低的对KRAS基因消融的敏感性。了解表观遗传开关和QM-PDA中异常蛋白质合成的需要将使我们能够更好地了解这种高度侵袭性的PDA亚型。本研究结果可作为研究新药和治疗方案的基础，以增加这种致命疾病的治疗机会。

Pancreatic Ductal Adenocarcinoma (PDA) is an extremely lethal disease with a 5-year survival rate of less than 10% and is likely to become the second leading cause of cancer related deaths by 2030. We know that pancreatic cancer is complex given that patients respond very differently to chemotherapy. To understand these differences, researchers have recently characterized two main subtypes: classical/pancreatic progenitor and quasi-mesenchymal (QM)/basal/squamous. The QM PDA subtype is more aggressive and has the worst overall survival. Understanding what defines each subtype, their susceptibilities and mechanisms of resistance will help to identify new targeted therapies for this devastating disease. While investigating these questions the Kugel lab made the startling discovery that the QM PDA subtype expresses high levels of high-mobility group (HMG) protein A2 (HMGA2) and have evolved mechanisms to dramatically increase protein translation. This raised the intriguing possibility that the aberrant activation of HMGA2 increases protein translation and may be one mechanism by which PDA attains the highly aggressive QM phenotype. The aim of this proposal is to elucidate the role of HMGA2 in regulating tDNA transcription in QM PDA, determine the role of differentially expressed tRNAs on protein synthesis and finally, explore the role of HMGA2 in PDA subtype determination, initiation and maintenance. Therefore, the Cancer Genome Atlas will be utilized to investigate potential candidate tRNAs, that will be validated in HMGA2 (HMGA2high/QM PDA) cell lines against HMGA2low/classical PDA cell lines. The direct influence of HMGA2 on tRNA expression will be tested in classical or QM PDA cell lines engineered to either overexpress WT HMGA2 or silence HMGA2 by siRNA, respectively by tRNA sequencing. HMGA2 acts as architectural transcription factor. We will assay the effect of HMGA2 on chromatin structure to induce tDNA transcription by ChIP analysis of canonical and non-canonical histones bound to candidate tRNA gene bodies in engineered PDA cell lines. We will perform ribosome profiling to identify changes in the protein synthesis due to differentially expressed tRNAs. Lastly, the engineered cell lines will be used to evaluate the effect of HMGA2 on QM-markers like increased TP63DeltaN and mesenchymal markers expression, increased chromatin bound MYC levels and reduced sensitivity to genetic ablation of KRAS. Understanding the epigenetic switch and the need of aberrant protein synthesis in QM-PDA will allow us to better understand this highly aggressive PDA subtype. The findings of this proposal could be the base to investigate into new drugs and therapeutic plans to increase the therapeutic chances of this lethal disease.